Therapeutic protocols used today by medical practitioners in treatment of their patient population requires accurate and convenient methods of monitoring patient disease states. Much effort has been directed to research and development of methods for measuring the presence and/or concentration of biologically significant substances indicative of a clinical condition or disease state, particularly in body fluids such as blood, urine or saliva. Such methods have been developed to detect the existence or severity of a wide variety of disease states such as diabetes, metabolic disorders, hormonal disorders, and for monitoring the presence and/or concentration of ethical or illegal drugs. More recently there have been significant advancements in the use of affinity-based electrochemical detection/measurement techniques which rely, at least in part, on the formation of a complex between the chemical species being assayed (the "analyte") and another species to which it will bind specifically (a "specific binding partner"). Such methods typically employ a labeled ligand analog of the target analyte, the ligand analog selected so that it binds competitively with the analyte to the specific binding partner. The ligand analog is labeled so that the extent of binding of the labeled ligand analog with the specific binding partner can be measured and correlated with the presence and/or concentration of the target analyte in the biological sample.
Numerous labels have been employed in such affinity based sample analysis techniques, including enzyme labeling, radioisotopic labeling, fluorescent labeling, and labeling with chemical species subject to electrochemical oxidation and/or reduction. The use of redox reversible species, sometimes referred to as electron transfer agents or electron mediators as labels for ligand analogs, have proven to provide a practical and dependable results in affinity-based electrochemical assays. However, the use of electrochemical techniques in detecting and quantifying concentrations of such redox reversible species (correlating with analyte concentrations) is not without problem. Electrochemical measurements are subject to many influences that affect the accuracy of the measurements, including those relating to variations in the electrode structure itself and/or matrix effects deriving from variability in liquid samples.
The present invention relates to immunosensors based on direct electrochemical measurement of detectable species with microarray electrodes under bipotentiostatic control. An electrochemical label, for example an Os mediator, is covalently attached to a peptide which has amino acid sequence of the binding epitope for the antibody. When indicator/peptide conjugate is bound to antibody, the indicator does not function electrochemically or it is said to be "inhibited". The analyte present in sample will compete with indicator/peptide conjugate for the limited number of binding sites on the antibody. When more analyte is present, more free indicator/peptide conjugate will be left producing higher current at a sensor electrode, i.e., one of the working electrodes where measured events (oxidation or reduction) are taking place. In the opposite case, when less analyte is present, more indicator/peptide conjugate will be bound to antibody resulting less free conjugates and producing lower current levels at the working electrodes. Therefore the current detected at either one of the working electrodes will be a function of analyte concentration.
It is frequently desired to measure more than one analyte species in a liquid sample. Measurement of multiple species in a mixture has been achieved with photometry and fluorescence, via selection of the appropriate wavelengths. Electrochemical measurements of a single species in a complex mixture are routinely made by selecting a potential at which only the desire species is oxidized or reduced (amperometry) or by stepping or varying the potential over a range in which only the desired species changes its electrochemical properties (AC and pulse methods). These methods suffer from disadvantages including lack of sensitivity and lack of specificity, interference by charging and matrix polarization currents (pulse methods) and electrode fouling due to the inability to apply an adequate overpotential. Moreover, electrochemical measurements are complicated by interference between the multiplicity of electroactive species commonly extant in biological samples.
Electrode structures which generate steady state current via diffusional feedback, including interdigitated array electrodes (IDAs) (FIGS. 1 and 2) and parallel plate arrangements with bipotentiostatic control are known. They have been used to measure reversible species based on the steady state current achieved by cycling of the reversible species. A reversible mediator (redox reversible species) is alternately oxidized and reduced on the interdigitated electrode fingers. The steady state current is proportionate to mediator concentration (FIG. 3) and limited by mediator diffusion. A steady state current is achieved within seconds of applying the predetermined anodic (more positive) and cathodic (less positive or negative) potentials (FIG. 6) to the microelectrode array. The slope of a plot of the IDA current vs. mediator concentration is dependent on IDA dimensions, and the slope increases with narrower electrode spacings (FIG. 7).
One embodiment of the present invention provides a method for measuring multiple analyte species in the same sample, and optimally on the same electrode structure, thus improving the accuracy of the relative measurements. This invention also provides an electrochemical biosensor with capacity to provide improved accuracy through the use of self-compensation. Analyte concentration can be measured/calculated from electrometric data obtained on the same liquid sample with the same electrode structure (the working electrodes), thereby minimizing perturbations due to variability in sample or electrode structure.
The various embodiments of this invention utilize the principle of diffusional recycling, where a diffusible redox reversible species is alternately oxidized and reduced at nearby electrodes, thereby generating a measurable current. As alternate oxidation and reduction is required for measurement, only electroactive species which are electrochemically reversible are measured thereby eliminating, or at least reducing, the impact or interference from non-reversible electroactive species in the sample. Redox reversible species having different oxidation potentials can be independently measured in a mixture by selecting and bipotentiostatically controlling the oxidizing and reducing potentials for neighboring electrode pairs so that only the species of interest is oxidized at the anode (the electrode with the more positive potential) and reduced at the cathode (the electrode with the less positive or negative potential). When the working electrodes (the anode/cathode arrays) are dimensioned to allow diffusional recycling of the redox-reversible-species at the selected oxidizing and reducing potentials appropriate for that species, a steady state current at the working electrodes where the measurable oxidative and reductive events are taking place, is quickly established through the sample and the electrode structure. The magnitude of the current is proportional to the concentration of the diffusible redox reversible species in the sample. When two or more redox reversible species are utilized, they are selected to have redox potentials differing by at least 50 millivolts, most preferably at least 200 millivolts, to minimize interference between one species and the other in measurements of the respective steady state currents.
Any electrode structure which allows for diffusional recycling to achieve steady state current in response to application of pre-selected species-specific anodic and cathodic potentials can be utilized in carrying out the invention. Suitable electrode structures include interdigitated array microelectrodes and parallel plate electrodes separated by distances within the diffusion distance of the respective redox reversible species. The electrode structures typically include a reference electrode (e.g., Ag/AgCl), at least two working electrodes (one at positive potential and another at a less positive or negative potential relative to the reference electrode), and optionally an auxiliary electrode for current control. In use, a programmable bipotentiostat is placed in electrical communication with the electrode structure for applying the respective anodic and cathodic potentials specific for each of the respective redox reversible species utilized in the method/biosensor. Several novel osmium complexes have been developed for use as labels for preparing ligand analog conjugates having potential differences sufficient to allow the use of two osmium complexes (as opposed to an osmium complex and a ferrocene or other redox reversible label) in this invention.
Accordingly, one embodiment of the invention provides a device for detecting or quantifying one or more analytes in a liquid sample. The device comprises at least two redox reversible species having respective redox potentials differing by at least 50 millivolts, and an electrode structure for contact with the liquid sample. In one embodiment the device further comprises a chamber for containing the liquid sample, optionally dimensioned for capillary fill. The electrode structure includes a reference electrode and an anode and a cathode (working electrodes) dimensioned to allow diffusional recycling of the redox reversible species in the sample when a redox-reversible-species-dependent cathodic potential is applied to one working electrode and a redox-reversible-species-dependent anodic potential is applied to a second electrode to enable and sustain a measurable current through the sample. The device also includes conductors communicating with the respective electrodes for applying potentials and for carrying current conducted between the sample and the respective electrodes.
The device in accordance with this invention is typically utilized in combination with a meter which includes a power source, for example a battery, a microprocessor, a register for storing measured current values, and a display for reporting calculated analyte concentrations based on measured current values. The construction and configuration of such meters are well known in the art. Meters for use in accordance with the present device further comprise a bipotentiostat under control of the microprocessor or separately programmable to apply predetermined potentials to the device component microelectrode arrays during liquid sample analysis. Improvements in meter construction and design for biosensor systems are described in U.S. Pat. Nos. 4,999,632; 5,243,516; 5,366,609; 5,352,351; 5,405,511; and 5,48,271, the disclosures of which are hereby incorporated by reference.
In another embodiment of the invention there is provided a method for measuring the concentration of one or more analytes in a liquid sample. The method includes contacting a portion of the sample with pre-determined amounts of at least a first and second redox reversible species having a redox potential differing by at least 50 millivolts from that of each other species. Each respective species comprises a liquid sample diffusible conjugate of a ligand analog of an analyte in the liquid sample and a redox reversible label. The liquid sample is also contacted with a predetermined amount of at least one specific binding partner for each analyte to be measured. The diffusible conjugate is selected so that it is capable of competitive binding with the specific binding partner for said analyte.
The concentration of diffusible redox-reversible-species in the liquid sample is then determined electrochemically. The sample is contacted with an electrode structure, including a reference electrode and at least first and second working electrodes dimensioned to allow diffusional recycling of at least one of the diffusible redox-reversible-species in the sample, when a predetermined redox-reversible-species-dependent cathodic potential is applied to one working electrode and a predetermined redox-reversible-species-dependent anodic potential is applied to the second working electrode. Typically, a first cathodic potential is applied to the first working electrode and a first anodic potential is applied to the second working electrode to establish current flow through the sample due to diffusional recycling of the first redox-reversible-species without significant interference from the second redox-reversible-species. Current flow through one or more of the electrodes at the first anodic and cathodic potentials is measured. Similarly current flow responsive to application of second cathodic and anodic potentials to electrodes in contact with the sample is measured and correlated with measured current flows for known concentrations of the respective redox-reversible-species, said concentrations being proportionate to the respective analyte concentrations at a predetermined redox-reversible species-dependent potential (anodic or cathodic). Alternatively, the potential of one of the working electrodes can be held constant and current flow is monitored as the potential of the other working electrode is varied and swept through the other redox-reversible species-dependent potential.
The reagent components for the invention, including the redox reversible species and the specific binding partners, can be provided in the form of a test kit for measuring the targeted analyte(s) in a liquid sample, either as separate reagents or, more preferably, combined as a multi-reagent composition, e.g. combined redox reversible species, combined specific binding partners, or combined redox reversible species and specific binding partners. The kit optionally, but preferably, includes an electrode structure dimensioned to allow diffusional redox recycling of diffusible redox reversible species in the liquid sample. The electrode structure includes conductors for connecting the structure bipotentiostat programmed to apply redox-reversible-species-dependent-anodic and cathodic potentials to the electrode structure and to sense and measure current flow, typically at one or both of the working electrodes, responsive to such applied potentials.
Also described herein is the preparation and use of electrochemically detectable osmium complexes and covalent conjugates of said complexes having oxidation potentials differing sufficiently to enable their use together in the respective method and device embodiments of the invention. Osmium labeled ligand analogs capable of binding to a specific binding partner of a biologically significant analyte are prepared. One group of electrochemically detectable conjugates comprise a bis(bipyridyl) imidazolyl chloroosmonium complex characterized by fast mediation kinetics and low redox potential (+15 mV vs. Ag/AgCl). Another group of osmium complex labeled, electrochemically detectable conjugates include tris(bipyridyl) osmium complexes, which, like the bis(bipyridyl) imidazolyl chloroosmium complexes are characterized by fast mediation kinetics, but the tris(bipyridyl) complexes have a redox potential sufficiently different from the bis(pyridyl) imidazolyl chloroosmium complexes to allow their use together in the various embodiments of this invention to enable use of microelectrode arrays for measuring more than one analyte in a single liquid sample by concentration dependent currents amplified by diffusional redox recycling.
In one preferred embodiment of the invention at least one osmium complex conjugates is used in combination with another conjugated redox-reversible-species for the measurement of both glycosylated hemoglobin and hemoglobin in a lysed blood sample. One redox-reversible-species preferably comprises an osmium complex covalently linked to a ligand analog of either hemoglobin or glycosylated hemoglobin, and the second redox-reversible-species comprises a second redox reversible label covalently bound to a ligand analog of the other of the two target analytes. The method enables measurement of the concentration of both the glycosylated hemoglobin (HbAlc) and the concentration of either total hemoglobin or that of unglycosylated hemoglobin (HbA.sub.0) thereby enabling calculation of the results as a ratio of the two measurements (% HbAlc). It is advantageous to assay both HbAlc and total hemoglobin (or HbA.sub.0) using the same principle in a single sample.